Study on in vitro isolation and culture method of neural stem cells from fetal rat neocortex

Objective To establish method for isolation and culture of neural stem cells from fetal rat neocortex in vitro.Methods Neocortexes of fetal SD rats were isolated on gestational day 14.Mechanical triturations were used to improve cell dissociation status and conditions of serum-free media were optimized.Three test methods including neural stem cell marker protein(nestin and SOX2),proliferation and clonogenic ability and the multilineage differentiation potential were employed to identify neural stem cells.Results Nestin of cells cultured with this method was expressed with strong positive,and more than 99% cells were SOX2-positive.BrdU was incorporated into the cells,and the amounts of the cells were 10.55 times as much as the initial cell number after 3 days of culture.The ratios of clonal neurosphere were(33.00 ± 4.40)% after 6 days of culture.Staining with specific protein markers in the corresponding cells confirmed that neural stem cells could form neurons(MAP2),astrocytes(GFAP) and oligodendrocytes(O4) after differentiation induction.Conclusion Neural stem cells obtained with this method had high purity and cell output,potent self-renewal and multi-lineage differentiation ability,which will provide a good model for toxicological research.